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Powdery mildew (PM) is a severe fungal disease of cucumber worldwide. Identification of genetic factors resistant to PM is of great importance for marker-assisted breeding to ensure cucumber production. Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) have been shown to play important roles in plant development and immunity; however, whether they have a role in PM response in cucurbit crops remains unknown. We performed strand-specific RNA sequencing and miRNA sequencing using RNA from cucumber leaves of two near-isogenic lines (NILs), S1003 and NIL (Pm5.1) infected with PM, and systematically characterized the profiles of cucumber lncRNAs and messenger RNA (mRNAs) responsive to PM. In total, we identified 12,903 lncRNAs and 25,598 mRNAs responsive to PM. Differential expression (DE) analysis showed that 119 lncRNAs and 136 mRNAs correlated with PM resistance. Functional analysis of these DE lncRNAs and DE mRNAs revealed that they are significantly associated with phenylpropanoid biosynthesis, phenylalanine metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and endocytosis. Particularly, two lncRNAs, LNC_006805 and LNC_012667, might play important roles in PM resistance. In addition, we also predicted mature miRNAs and competing endogenous RNA (ceRNA) networks of lncRNA–miRNA–mRNA involved in PM resistance. A total of 49 DE lncRNAs could potentially act as target mimics for 106 miRNAs. Taken together, our results provide an abundant resource for further exploration of cucumber lncRNAs, mRNAs, miRNAs, and ceRNAs in PM resistance, and will facilitate the molecular breeding for PM-resistant varieties to control this severe disease in cucumber.

BackgroundTrichomes are excellent model systems for the analysis of cell differentiation and play essential roles in plant protection. From cucumber inbred line ‘WD1’, we identified an EMS-induced trichome abnormally developing mutant,nps, which exhibited smaller, denser and no pyramid-shaped head trichomes.ResultsUsing F2and BC1populations constructed from a cross betweennpsand ‘9930’, the genetic analysis showed that thenpstrait is controlled by a single recessive nuclear gene. We identifiedCsNpsby map-based cloning with 576 individuals of the F2population generated from the cross ofnpsand inbred line ‘9930’. TheCsNpswas located at a 13.4-kb genomic region on chromosome 3, which region contains three predicted genes. Sequence analysis showed that only one single nucleotide mutation (C → T) between 9930 andnpswas found in the second exon ofCsa3G748220, a plant-specific class I HD-Zip gene. The result of allelism test also indicated thatnpsis a novel allelic mutant ofMict(Micro-trichome). Thus,npswas renamedmict-L130F. By comparing the transcriptome ofmict-L130Fvs WD1 and 06–2 (mict) vs 06–1 (wildtype, near-isogenic line of 06–2), several potential target genes that may be related to trichome development were identified.ConclusionsOur results demonstrate thatMict-L130Fis involved in the morphogenesis of trichomes. Map-based cloning of theMict-L130Fgene could promote the study of trichome development in cucumber.

An unusual flower and tendril (uft) mutant in cucumber was caused by a mutation in Csa1G056950 encoding an F-box protein. Flowers and tendrils are important agronomic and yield traits of cucumber (Cucumis sativus L.). In this study, we identified an unusual flower and tendril (uft) mutant from an ethyl methanesulfonate (EMS) mutagenesis population. Genetic analysis revealed that the phenotype of the uft mutant was regulated by a single recessive nuclear gene. Map-based cloning and MutMap+ results demonstrated that Csa1G056950 (CsUFO), encoding an F-box protein, was the causal gene for the uft mutant phenotype of cucumber. A single nucleotide polymorphism (SNP) mutation (C to T) in the second exon of CsUFO resulted in premature translation termination. The expression level of CsUFO was significantly decreased in apical buds of the uft mutant compared with the wild-type (WT) WD1. Transcriptome analysis indicated that many genes for organ development were down-regulated in uft plants, suggesting CsUFO-associated networks that regulate flower and tendril development. These findings provide a new insight into understanding the molecular mechanisms of flower organogenesis in cucumber.

CsSh5.1, which controls hypocotyl elongation under high temperature conditions in cucumber, was mapped to a 57.1 kb region on chromosome 5 containing a candidate gene encoding a xyloglucan galactosyltransferase. Hypocotyl growth is a vital process in seedling establishment. Hypocotyl elongation after germination relies more on longitudinal cell elongation than cell division. Cell elongation is largely determined by the extensibility of the cell wall. Here, we identified a spontaneous mutant in cucumber (Cucumis sativus L.), sh5.1, which exhibits a temperature-insensitive short hypocotyl phenotype. Genetic analysis showed that the phenotype of sh5.1 was controlled by a recessive nuclear gene. CsSh5.1 was mapped to a 57.1 kb interval on chromosome 5, containing eight predicted genes. Sequencing analysis revealed that the Csa5G171710 is the candidate gene of CsSh5.1, which was further confirmed via co-segregation analysis and genomic DNA sequencing in natural cucumber variations. The result indicated that hypocotyl elongation might be controlled by this gene. CsSh5.1 encodes a xyloglucan galactosyltransferase that specifically adds galactose to xyloglucan and forms galactosylated xyloglucans, which determine the strength and extensibility of the cell walls. CsSh5.1 expression in wild-type (WT) hypocotyl was significantly higher than that in sh5.1 hypocotyl under high temperature, suggesting its important role in hypocotyl cell elongation under high temperature. The identification of CsSh5.1 is helpful for elucidating the function of xyloglucan galactosyltransferase in cell wall expansion and understanding the mechanism of hypocotyl elongation in cucumber.

Plants monitor environmental cues to balance growth by optimizing their inflorescence architecture. TERMINAL FLOWER 1 (TFL1) and its orthologues regulate the inflorescence structure in cucumber, yet the mechanisms underlying their responses to environmental factors and the formation of terminal flowers remain elusive. Here, we performed map-based cloning to identify the gene that controls a season-dependent determinate growth phenotype and found that it was caused by the complete deletion of CsTFL1 in the genome of cucumber line WI1983Hde. In the CsTFL1 deletion plants (CsTFL1del ), determinate growth could be partially rescued by high-temperature and long-day conditions. The expressions of CsTFL1 and its orthologue CsTFL1d could be upregulated by long-day and high-temperature signals. Knockdown of CsTFL1d resulted in determinate growth and the formation of terminal flowers in WT. These results indicate that the induction of CsTFL1d expression by long-day and high- temperature might partially rescue determinate growth of CsTFL1del . Furthermore, biochemical analyses showed that CsTFL1d interacts directly with CsNOT2a, which indicated that CsTFL1d and CsTFL1 function via similar regulatory mechanism. Our data suggest that CsTFL1 and CsTFL1d co-contribute to inhibit determinate growth by responding to temperature and photoperiod signals. It provides mechanistic insights into how environmental cues sculpt the inflorescence architecture of cucumber. This article is protected by copyright. All rights reserved.

Unisexual flowers provide a useful system for studying plant sex determination. In cucumber (Cucumis sativus L.), three major Mendelian loci control unisexual flower development, Female (F), androecious [a; 1-aminocyclopropane-1-carboxylate {ACC} synthase 11, acs11], and Monoecious (M; ACS2), referred to here as the Female, Androecious, Monoecious (FAM) model, in combination with two genes, gynoecious (g, the WIP family C2H2 zinc finger transcription factor gene WIP1) and the ethylene biosynthetic gene ACC oxidase 2 (ACO2). The F locus, conferring gynoecy and the potential for increasing fruit yield, is defined by a 30.2-kb tandem duplication containing three genes. However, the gene that determines the Female phenotype, and its mechanism, remains unknown. Here, we created a set of mutants and revealed that ACS1G is responsible for gynoecy conferred by the F locus. The duplication resulted in ACS1G acquiring a new promoter and expression pattern; in plants carrying the F locus duplication, ACS1G is expressed early in floral bud development, where it functions with ACO2 to generate an ethylene burst. The resulting ethylene represses WIP1 and activates ACS2 to initiate gynoecy. This early ACS1G expression bypasses the need for ACS11 to produce ethylene, thereby establishing a dominant pathway for female floral development. Based on these findings, we propose a model for how these ethylene biosynthesis genes cooperate to control unisexual flower development in cucumber.

The leaf is an important photosynthetic organ and plays an essential role in the growth and development of plants. Leaf color mutants are ideal materials for studying chlorophyll metabolism, chloroplast development, and photosynthesis. In this study, we identified an EMS-induced mutant, yl2.1, which exhibited yellow cotyledons and true leaves that did not turn green with leaf growth. The yl2.1 locus was controlled by a recessive nuclear gene. The CsYL2.1 was mapped to a 166.7-kb genomic region on chromosome 2, which contains 24 predicted genes. Only one non-synonymous single nucleotide polymorphism (SNP) was found between yl2.1 and wt-WD1 that was located in Exon 7 of Csa2G263900, resulting in an amino acid substitution. CsYL2.1 encodes a plastid isoform of triose phosphate isomerase (pdTPI), which catalyzes the reversible conversion of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (GAP) in chloroplasts. CsYL2.1 was highly expressed in the cotyledons and leaves. The mesophyll cells of the yl2.1 leaves contained reduced chlorophyll and abnormal chloroplasts. Correspondingly, the photosynthetic efficiency of the yl2.1 leaves was impaired. Identification of CsYL2.1 is helpful in elucidating the function of ptTPI in the chlorophyll metabolism and chloroplast development and understanding the molecular mechanism of this leaf color variant in cucumber.

TCP proteins are plant-specific transcription factors widely implicated in leaf morphogenesis and senescence, flowering, lateral branching, hormone crosstalk, and stress responses. However, the relationship between the transcription pattern of TCPs and organ development in cucumber has not been systematically studied. In this study, we performed a genome-wide identification of putative TCP genes and analyzed their chromosomal location, gene structure, conserved motif, and transcript expression. A total of 27 putative TCP genes were identified and characterized in cucumber. All 27 putative CsTCP genes were classified into class I and class II. Class I comprised 12 CsTCPs and Class II contained 15 CsTCPs. The 27 putative CsTCP genes were randomly distributed in five of seven chromosomes in cucumber. Four putative CsTCP genes were found to contain putative miR319 target sites. Quantitative RT-PCR revealed that 27 putative CsTCP genes exhibited different expression patterns in cucumber tissues and floral organ development. Transcript expression and phenotype analysis showed that the putative CsTCP genes responded to temperature and photoperiod and were induced by gibberellin (GA)and ethylene treatment, which suggested that CsTCP genes may regulate the lateral branching by involving in multiple signal pathways. These results lay the foundation for studying the function of cucumber TCP genes in the future.

Trichomes and fruit spines are important traits that directly affect the appearance quality and commercial value of cucumber (Cucumis sativus). Tril (Trichome-less), encodes a HD-Zip IV transcription factor that plays a crucial role in the initiation of trichomes and fruit spines, but little is known about the details of the regulatory mechanisms involved. In this study, analysis of tissue expression patterns indicated that Tril is expressed and functions in the early stages of organ initiation and development. Expression of Tril under the control of its own promoter (the TrilPro::Tril-3*flag fragment) could partly rescue the mutant phenotypes of tril, csgl3 (cucumber glabrous 3, an allelic mutant of tril), and fs1 (few spines 1, a fragment substitution in the Tril promoter region), providing further evidence that Tril is responsible for the initiation of trichomes and fruit spines. In lines with dense spine, fs1-type lines, and transgenic lines of different backgrounds containing the TrilPro::Tril-3*flag foreign fragment, spine density increased in conjunction with increases in Tril expression, indicating that Tril has a gene dosage effect on fruit spine density in cucumber. Numerous Spines (NS) is a negative regulatory factor of fruit spine density. Characterization of the molecular and genetic interaction between Tril and NS/ns demonstrated that Tril functions upstream of NS with respect to spine initiation. Overall, our results reveal a novel regulatory mechanism governing the effect of Tril on fruit spine development, and provide a reference for future work on breeding for physical quality in cucumber.

Using map-based cloning of ts gene, we identified a new sort of gene involved in the initiation of multicellular tender spine in cucumber. The cucumber (Cucumis sativus L.) fruit contains spines on the surface, which is an extremely valuable quality trait affecting the selection of customers. In this study, we elaborated cucumber line NC072 with wild type (WT) hard fruit spines and its spontaneous mutant NC073, possessing tender and soft spines on fruits. The mutant trait was named as tender spines (ts), which is controlled by a single recessive nuclear gene. We identified the gene ts by map-based cloning with an F2 segregating population of 721 individuals generated from NC073 and WT line SA419-2. It was located between two markers Indel6239679 and Indel6349344, 109.7 kb physical distance on chromosome 1 containing fifteen putative genes. With sequencing and quantitative reverse transcription-polymerase chain reaction analysis, the Csa1G056960 gene was considered as the most possible candidate gene of ts. In the mutant, Csa1G056960 has a nucleotide change in the 5′ splicing site of the second intron, which causes different splicing to delete the second exon, resulting in a N-terminal deletion in the predicted amino acid sequence. The gene encodes a C-type lectin receptor-like tyrosine-protein kinase which would play an important role in the formation of cucumber fruit. This is firstly reported of a receptor kinase gene regulating the development of multicellular spines/trichomes in plants. The ts allele could accelerate the molecular breeding of cucumber soft spines.

Cucumber is one of the most important vegetables in the world. The C2H2 zinc finger protein (C2H2-ZFP) family plays an important role in the growth development and abiotic stress responses of plants. However, there have been no systematic studies on cucumber. In this study, we performed a genome-wide study of C2H2-ZFP genes and analyzed their chromosomal location, gene structure, conservation motif, and transcriptional expression. In total, 101 putative cucumber C2H2-ZFP genes were identified and divided into six groups (I–VI). RNA-seq transcriptome data on different organs revealed temporal and spatial expression specificity of the C2H2-ZFP genes. Expression analysis of sixteen selected C2H2-ZFP genes in response to cold, drought, salt, and abscisic acid (ABA) treatments by real-time quantitative polymerase chain reaction showed that C2H2-ZFP genes may be involved in different signaling pathways. These results provide valuable information for studying the function of cucumber C2H2-ZFP genes in the future.

Background Pedicel orientation can affect the female flower orientation and seed yield in cucumber. A spontaneous mutant possessing upward growth of pedicels was identified in the wild type inbred strain 9930 and named upward-pedicel (up). The morphological and genetic analyses of up were performed in this study. In order to clone the up gene, 933 F2 individuals and 524 BC1 individuals derived from C-8-6 (WT) and up were used for map-based cloning. Results up was mapped to a 35.2 kb physical interval on chromosome 1, which contains three predicted genes. Sequencing analysis revealed that a 5-bp deletion was found in the second exon of Csa1G535800, and it led to a frameshift mutation resulting in a premature stop codon. The candidate gene of CsUp (Csa1G535800) was further confirmed via genomic and cDNA sequencing in biparental and natural cucumber populations. Sequencing data showed that a 4-bp deletion was found in the sixth exon of Csa1G535800 in CGN19839, another inbred line, and there was also a mutation of an amino acid in Csa1G535800 that could contribute to the upward growth of pedicels in CGN19839. Moreover, it was found that Csa1G535800 exhibited strong expression in the pedicel of WT, suggesting its important role in development of pedicel orientation. Thus, Csa1G535800 was considered to be the candidate gene of CsUp. Conclusions CsUp encodes an Auxilin-like protein and controls pedicel orientation in cucumber. The identification of CsUp may help us to understand the mechanism of pedicel orientation development and allow for investigation of novel functions of Auxilin-like proteins in cucumber.

The regulatory gene network of unicellular trichome development in Arabidopsis thaliana has been studied intensively, but that of multicellular remains unclear. In the present study, we characterized cucumber trichomes as representative multicellular and unbranched structures, but in a spontaneous mutant, mict (micro-trichome), all trichomes showed a micro-size and stunted morphologies. We revealed the transcriptome profile using Illumina HiSeq 2000 sequencing technology, and determined that a total of 1391 genes exhibited differential expression. We further validated the accuracy of the transcriptome data by RT-qPCR and found that 43 genes encoding critical transcription factors were likely involved in multicellular trichome development. These 43 candidate genes were subdivided into seven groups: homeodomain, MYB-domain, WRKY-domain, bHLH-domain, ethylene-responsive, zinc finger and other transcription factor genes. Our findings also serve as a powerful tool to further study the relevant molecular networks, and provide a new perspective for investigating this complex and species-specific developmental process.

Structural variations (SVs) represent a major source of genetic diversity. However, the functional impact and formation mechanisms of SVs in plant genomes remain largely unexplored. Here, we report a nucleotide-resolution SV map of cucumber (Cucumis sativas) that comprises 26,788 SVs based on deep resequencing of 115 diverse accessions. The largest proportion of cucumber SVs was formed through nonhomologous end-joining rearrangements, and the occurrence of SVs is closely associated with regions of high nucleotide diversity. These SVs affect the coding regions of 1676 genes, some of which are associated with cucumber domestication. Based on the map, we discovered a copy number variation (CNV) involving four genes that defines the Female (F) locus and gives rise to gynoecious cucumber plants, which bear only female flowers and set fruit at almost every node. The CNV arose from a recent 30.2-kb duplication at a meiotically unstable region, likely via microhomology-mediated break-induced replication. The SV set provides a snapshot of structural variations in plants and will serve as an important resource for exploring genes underlying key traits and for facilitating practical breeding in cucumber.

Cucumber fruits that have tubercules and spines (trichomes) are known to possess a warty (Wty) phenotype. In this study, the tuberculate fruit gene Tu was identified by map-based cloning, and was found to encode a transcription factor (TF) with a single C2 H2 zinc finger domain. Tu was identified in all 38 Wty lines examined, and was completely absent from all 56 non-warty (nWty) lines. Cucumber plants transgenic for Tu (TCP) revealed that Tu was required for the Wty fruit phenotype. Subcellular localization showed that the fusion protein GFP-Tu was localized mainly to the nucleus. Based on analyses of semi-quantitative and quantitative reverse transcription polymerase chain reaction (RT-PCR), and mRNA in situ hybridization, we found that Tu was expressed specifically in fruit spine cells during development of fruit tubercules. Moreover, cytokinin (CTK) content measurements and cytological observations in Wty and nWty fruits revealed that the Wty fruit phenotype correlated with high endogenous CTK concentrations. As a result of further analyses on the transcriptomic profile of the nWty fruit epidermis and TCP fruit warts, expression of CTK-associated genes, and hormone content in nWty fruit epidermis, Wty fruit warts and epidermis, and TCP fruit warts and epidermis, we found that Tu probably promoted CTK biosynthesis in fruit warts. Here we show that Tu could not be expressed in the glabrous and tubercule-free mutant line gl that contained Tu, this result that futher confirmed the epistatic effect of the trichome (spine) gene Gl over Tu. Taken together, these data led us to propose a genetic pathway for the Wty fruit trait that could guide future mechanistic studies.

It is well established that the plant hormone ethylene plays a key role in cucumber sex determination. Since the unisexual control gene M was cloned and shown to encode an ethylene synthase, instead of an ethylene receptor, the 'one-hormone hypothesis', which was used to explain the cucumber sex phenotype, has been challenged. Here, the physiological function of CsACS2 (the gene encoded by the M locus) was studied using the transgenic tobacco system. The results indicated that overexpression of CsACS2 increased ethylene production in the tobacco plant, and the native cucumber promoter had no activity in transgenic tobacco (PM). However, when PM plants were treated with exogenous ethylene, CsACS2 expression could be detected. In cucumber, ethylene treatment could also induce transcription of CsACS2, while inhibition of ethylene action reduced the expression level. These findings suggest a positive feedback regulation mechanism for CsACS2, and a modified 'one-hormone hypothesis' for sex determination in cucumber is proposed.

The high-density consensus map was constructed based on the GY14 × PI 183967 map from an inter-subspecific cross and the extended S94 × S06 map from an intra-subspecific cross. The consensus map was composed of 1,369 loci, including 1,152 SSR loci, 192 SRAP loci, 21 SCAR loci and one STS locus as well as three gene loci of fruit external quality traits in seven chromosomes, and spanned 700.5 cM, of which 682.7 cM (97.5%) were covered by SSR markers. The average genetic distance and physical interval between loci were 0.51 cM and ~268 kbp, respectively. Additionally, the physical position of the sequence-associated markers aligned along the assembled cucumber genome sequence established a relationship between genetic maps and cucumber genome sequence and to a great extent validated the order of markers in individual maps and consensus map. This consensus map with a high marker density and well-ordered markers is a saturated and reliable linkage map for genetic analysis of cucumber or the Cucurbitaceae family of plants.

The cucumber lines, S94 (Northern China open-field type, powdery mildew (PM) susceptible) and S06 (European greenhouse type, PM resistant), and their F(6:7) populations were used to investigate PM resistance under seedling spray inoculation in 2005/Autumn and 2006/Spring. QTL analysis was undertaken based on a constructed molecular linkage map of the corresponding F(6) population using composite interval mapping. A total of four QTLs (pm1.1, pm2.1, pm4.1 and pm6.1) for PM resistance were identified and located on LG 1, 2, 4 and 6, respectively, explaining 5.2%-21.0% of the phenotypic variation. Three consistent QTLs (pm1.1, pm2.1 and pm4.1) were detected under the two test conditions. The QTL pm6.1 was only identified in 2005/Autumn. The total phenotypic variation explained by the QTLs was 52.0% and 42.0% in 2005/Autumn and 2006/Spring, respectively. Anchor markers tightly linked to those loci (

Using map-based cloning of Tril gene, we identified a homeodomain-leucine zipper gene involved in the initiation of multicellular trichomes (including the spines of fruit) in cucumber. Fruit spines are a special type of trichome that impacts the quality and appearance of cucumber (Cucumis sativus L.) fruit. Scanning electron microscopy revealed that the trichome-less (tril) mutant originating from European greenhouse cucumber has a completely glabrous phenotype on cotyledons, hypocotyls, young leaves, fruits, and fruit stalks. Genetic analysis revealed that tril was inherited as a recessive allele at a single locus. Using 1058 F2 individuals derived from a cross between cucumber tril mutant CGN19839 and the micro-trichome (mict) mutant 06-2, tril was mapped to chromosome 6, and narrowed down to a 37.4 kb genomic region which carries seven predicted genes. Genetic and molecular analyses revealed that gene Cucsa.045360 is a possible candidate gene for the differentiation of epidermal cells to trichomes. It is a member of the class IV homeodomain-leucine zipper (HD-Zip IV) family and encodes homeodomain and START domain, sharing 66.7 % predicted amino acid sequence identity to PROTODERMAL FACTOR2 (PDF2) and 35.0 % to GLABRA2 (GL2) of Arabidopsis. The homeobox domain had changed amino acid sequence because of an insertion in tril mutant. The results of genetic analysis and transcriptome profiling indicated that the Tril gene had an epistatic effect on the Mict gene in trichome development. Phenotypes of the tril mutant such as glabrous fruits and female flowers at every node could be used in developing new cultivars.

Using SRAP (sequence-related amplified polymorphism) markers a genetic linkage map of cucumber was constructed with a population consisting of 138 F2 individuals derived from a cross of the two cucumber lines, S06 and S52. In the survey of parental polymorphisms with 182 primer combinations, 64 polymorphism-revealing primer pairs were screened out, which generated totally 108 polymorphic bands with an average of 1.7 bands per primer pair and at most 6 bands from one primer pair. The constructed molecular linkage map included 92 loci, distributed in seven linkage groups and spanning 1164.2 cM in length with an average genetic distance of 12.6 cM between two neighboring loci. Based on this linkage map, the quantitative trait loci (QTL) for the lateral branch number (Ibn) and the lateral branch average length (Ibl) in cucumber were identified by QTLMapperl.6. A major QTL Ibn1 located between ME11 SA4B and ME5EM5 in LG2 could explain 10.63% of the total variation with its positively effecting allele from S06. A major QTL Ibl1 located between DC1OD3 and DC1EM14 in LG2 could account for 10.38% of the total variation with its positively effecting allele from S06.

Trichomes on plants, similar to fine hairs on animal and human bodies, play important roles in plant survival and development. They also represent a useful model for the study of cell differentiation. Although the regulatory gene network of unicellular trichome development in Arabidopsis thaliana has been well studied, the genes that regulate multicellular trichome development remain unclear. We confirmed that Cucumis sativus (cucumber) trichomes are multicellular and unbranched, but identified a spontaneous mutant, trichome-less (tril), which presented a completely glabrous phenotype. We compared the transcriptome profilings of the tril mutant and wild type using the Illumina HiSeq 2000 sequencing technology. A total of 991 genes exhibited differential expression: 518 were up-regulated and 473 were down-regulated. We further identified 62 differentially expressed genes that encoded crucial transcription factors and were subdivided into seven categories: homeodomain, MADS, MYB, and WRKY domains, ethylene-responsive, zinc finger, and other transcription factor genes. We further analyzed the tissue-expression profiles of two candidate genes, GLABRA2-like and ATHB51-like, using qRT-PCR and found that these two genes were specifically expressed in the epidermis and trichomes, respectively. These results and the tril mutant provide useful tools to study the molecular networks associated with multicellular trichome development.

Plant trichomes serve as a highly suitable model for investigating cell differentiation at the single-cell level. The regulatory genes involved in unicellular trichome development in Arabidopsis thaliana have been intensively studied, but genes regulating multicellular trichome development in plants remain unclear. Here, we characterized Cucumis sativus (cucumber) trichomes as representative multicellular and unbranched structures, and identified Micro-trichome (Mict), using map-based cloning in an F2 segregating population of 7,936 individuals generated from a spontaneous mict mutant. In mict plants, trichomes in both leaves and fruits, are small, poorly developed, and denser than in the wild type. Sequence analysis revealed that a 2,649-bp genomic deletion, spanning the first and second exons, occurred in a plant-specific class I homeodomain-leucine zipper gene. Tissue-specific expression analysis indicated that Mict is strongly expressed in the trichome cells. Transcriptome profiling identified potential targets of Mict including putative homologs of genes known in other systems to regulate trichome development, meristem determinacy, and hormone responsiveness. Phylogenic analysis charted the relationships among putative homologs in angiosperms. Our paper represents initial steps toward understanding the development of multicellular trichomes.

Sex determination in plants has various mechanism; however, a single gene locus controlling unisexual expression is unique in the Cucurbitaceae plants, particularly cucumber (Cucumis sativus L.) and melon (Cucumis melo L.). In this study, with quantitative RT-PCR method, two sets of near-isogenic lines (NILs) were used to analyze the expression pattern of gene CsACS2 (GenBank accession number FJ529216). Additionally, chemical applications (AgNO3 and AVG) were used to investigate the effect of the plant endogenous ethylene on CsACS2 expression. Expression analysis reveals that endogenous ethylene, which might be derived from F or M itself, could activate the expression of M gene.

Sex determination in plants involves a variety of mechanisms. Here, we report the map-based cloning and characterization of the unisexual-flower-controlling gene M. M was identified as a previously characterized putative 1-aminocyclopropane-1-carboxylic acid synthase gene, while the m allele that mutated at a conserved site (Gly33Cys) lost activity in the original enzymatically active allele.

Warty fruit is one of the highly valuable external quality traits related to the market values of cucumber. Genetic analysis has shown that a single dominant gene, Tu (Tuberculate fruit), determines the warty fruit trait in the cucumber plant. An F(2) population (247 individuals) from the cross of S06 x S52 was used for the mapping of the Tu/tu locus. By combining bulked segregant analysis with the sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) markers, 15 markers (9 SRAPs and 6 SSRs) linked to the Tu/tu locus were identified. Of nine SRAP markers, three closely linked to the Tu/tu locus were successfully converted into sequence characterized amplified region (SCAR) markers. The Tu/tu locus was mapped between the co-dominant SSR marker SSR16203 and the SCAR marker C_SC933, at a genetic distance of 1.4 and 5.9 cM, respectively. Then the linked SSR markers in the study were used as anchor loci to locate the Tu/tu locus on cucumber chromosome 5. Moreover, the validity analysis of the C_SC69 and C_SC24 markers was performed with 62 cucumber lines of diverse origins, showing that the two SCAR markers can be used for marker-assisted selection (MAS) of the warty fruit trait in cucumber breeding. The information provided in this study will facilitate the map-based cloning of the Tu/tu gene.

Owing to its diverse sex types, the cucumber plant has been studied widely as a model for sex determination. In addition to environmental factors and plant hormones, three major genes—F/f, M/m, and A/a—regulate the sex types in the cucumber plant. By combining the bulked segregant analysis (BSA) and the sequence-related amplified polymorphism (SRAP) technology, we identified eight markers linking to the M/m locus. Among them, the two closely linked SRAP markers flanking the M/m locus were the co-dominant marker ME1EM26 and the dominant marker ME1EM23. Further, the co-dominant marker ME8SA7 co-segregated with the M/m locus. With the chromosome walking method using the cucumber genomic bacterial artificial chromosome (BAC) library, we successfully developed a co-dominant SCAR marker S_ME1EM23 from the ME1EM23 sequence. Along with the other two co-dominant SCAR markers S_ME1EM26 and S_ME8SA7 (developed from ME1EM26 and ME8SA7, respectively) in a larger segregating population (900 individuals), the M/m locus was mapped between S_ME1EM26 (5.4 cM) and S_ME1EM23 (0.7 cM), and S_ME8SA7 co-segregated with it.

A group of 224 recombinant inbred lines (RILs) was derived from a narrow cross between 2 cucumber (Cucumis sativus L.) lines, namely, S94 (Northern China type with weak lateral branch growth potential and early lateral branch sprouting time) and S06 (Northern European type with strong lateral branch growth potential and late lateral branch sprouting time). These lines were then used for investigating lateral branch-related traits. A total of 36 quantitative trait loci (QTLs) were detected for the following 4 lateral branch-related traits: lateral branch average length (LBAL), lateral branch total length (LBTL), lateral branch number (LBN), and first lateral branch node (FLBN). Further, each QTL explained 3.1% (lbtl2.1, spring) to 32.3% (lbn2.3, spring) of the observed phenotypic variance. Eleven QTLs (lbal1.1, lbtl1.1, lbn1.2, flbn1.2, etc.) for different traits were found to be clustered on the e23m18d-ME23EM6c section (7.4 cM) of linkage group (LG) 1; further, 15 QTLs (lbal2.1, lbtl2.1, lbn2.1, flbn2.1, etc.) were found to be clustered on the S94A1-ME4SA4a section (13.9 cM) of LG2. Twenty-one QTLs explained more than 10% of the phenotypic variance. Moreover, lbtl1.3 (autumn, 26.2%, logarithm of odds (LOD) = 17.4; spring, 26.9%, LOD = 17.9) had stable position and contribution in both seasons. Several sequence-anchor markers (CMBR40, F, CS30, S94A1, CSWTA11B, etc.) were closely linked with some QTLs for LBAL, LBTL, LBN, and FLBN, which can be used for the marker-assisted selection to improve the plant architecture in cucumber breeding.